Carbonyl reductase, gene thereof and use of the same

ABSTRACT

The present invention provides a novel polypeptide forming (R)-2-chloro-1-(3′-chlorophenyl)ethanol, a polynucleotide coding for said polypeptide, and use of the same. 
     The present invention relates to a polypeptide having the following physical and chemical properties (1) to (4):
         (1) activity: acting on 2-chloro-1-(3′-chlorophenyl)ethanone with NADPH or NADH as a coenzyme, to form (R)-2-chloro-1-(31-chlorophenyl)ethanol;   (2) optimum pH for activity: 5.0 to 6.0;   (3) optimum temperature for activity: 40° C. to 50° C.;   (4) molecular weight: about 40,000 as determined by gel filtration analysis, about 30,000 as determined by SDS polyacrylamide gel electrophoresis analysis. The present invention also relates to a polypeptide comprising the amino acid sequence shown under SEQ ID NO:1 in the sequence listing, a polynucleotide coding for said polypeptide, and a transformant producing said polypeptide at high levels.

RELATED APPLICATIONS

This application is a nationalization of PCT Application No.PCT/JP03/05500 filed Apr. 30, 2003. This application claims priorityfrom Japanese Patent Application No. 2002-128648 filed on Apr. 30, 2002.

TECHNICAL FIELD

The present invention relates to a polypeptide having activity inasymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone representedby the formula (1):

to form (R)-2-chloro-1-(3′-chlorophenyl)ethanol represented by theformula (2):

as isolated from a microorganism having such activity, a polynucleotidecoding for said polypeptide, an expression vector containing saidpolynucleotide, and a transformant transformed with said expressionvector.

The present invention also relates to a method for producing opticallyactive alcohols, in particular optically active 1-phenylethanolderivatives or optically active 3-hydroxy ester derivatives using saidtransformant. Such optically active 1-phenylethanol derivatives oroptically active 3-hydroxy ester derivatives are compounds useful assynthetic materials for such as medicines and agricultural chemicals.

BACKGROUND ART

As for the methods for producing optically active 1-phenylethanolderivatives, there have been disclosed:

1) the method which comprises allowing a microorganism belonging to thegenus Ashbya or Ogataea or processed products thereof, for instance, toact on a 2-halo-1-(substituted phenyl)ethanone to form an opticallyactive 2-halo-1-(substituted phenyl)ethanol (Japanese Kokai PublicationHei-04-218384 and Japanese Kokai Publication Hei-11-215995), and

2) the method which comprises allowing dry cells of Geotrichum candidumto act on a 1-(substituted phenyl)ethanone to form an optically active1-(substituted phenyl)ethanol (J. Org. Chem., 63, 8957 (1998)).

As for the method for producing optically active 3-hydroxy esterderivatives, there has been disclosed:

3) the method which comprises allowing a recombinant Escherichia coli asobtained by introduction of a Sporobolomyces salmonicolor-derivedaldehyde reductase gene to act on a 4-substituted acetoacetic acid esterto form an (R)-4-substituted-3-hydroxybutyric acid ester (Japanese KokaiPublication Hei-08-103269).

However, all of these methods allow only a low substrate chargeconcentration or give a low rate of conversion from substrate toproduct. Thus, more efficient production method has been desired.

SUMMARY OF THE INVENTION

In view of the above-mentioned state of the art, the present inventionhas for its object to provide a polypeptide useful in the production ofoptically active 1-phenylethanol derivatives or optically active3-hydroxy ester derivatives, a polynucleotide coding for saidpolypeptide, an expression vector containing said polynucleotide, and atransformant transformed with said expression vector.

The present invention also has for its object to provide a method forefficiently producing optically active 1-phenylethanol derivatives oroptically active 3-hydroxy ester derivatives using said transformant.

The present inventors isolated a polypeptide having activity inasymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol, from a microorganism havingsuch activity, and found that use of said polypeptide make it possibleto efficiently produce not only (R)-2-chloro-1-(3′-chlorophenyl)ethanolbut also useful optically active alcohol, for example optically active1-phenylethanol derivatives, such as (S)-1-(2′-fluorophenyl)ethanol, andoptically active 3-hydroxy ester derivatives, typically ethyl(R)-4-chloro-3-hydroy butyrate. They also succeeded in isolating apolynucleotide coding for said polypeptide and further in creating anexpression vector and a transformant. Thus, the present invention hasbeen completed.

That is, the invention provides a polypeptide capable of asymmetricallyreducing 2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol.

The invention also provides a polynucleotide coding for the abovepolypeptide.

The invention further provides an expression vector containing the abovepolynucleotide.

The invention further provides a transformant capable of producing theabove polypeptide at high levels.

The invention still further provides a practical method for producingoptically active 1-phenylethanol derivatives or optically active3-hydroxy ester derivatives, typically(R)-2-chloro-1-(3′-chlorophenyl)ethanol, (S)-1-(2′-fluorophenyl)ethanoland ethyl (R)-4-chloro-3-hydroxybutyrate, using said transformant.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the invention is described in detail.

Usable as the polypeptide of the invention is any of those polypeptideshaving activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanbne represented by the formula (1):

to form (R)-2-chloro-1-(3′-chlorophenyl)ethanol represented by theformula (2):

Such a polypeptide can be isolated from a microorganism having theactivity mentioned above. The microorganism to be used as the source ofthe polypeptide is not particularly restricted but there may bementioned, for example, yeasts of the genus Rhodotorula, and Rhodotorulaglutinis var. dairenensis IFO 0415 is particularly preferred.

The microorganism producing the polypeptide of the invention may be awild strain or variant. A microorganism derived by a genetic engineeringtechnique, such as cell fusion or gene manipulation can be used as well.A genetically engineered microorganism producing the polypeptide of theinvention can be obtained, for example, by a method comprising the stepof isolating and/or purifying such polypeptide and determining a part orthe whole of the amino acid sequence thereof, the step of determiningthe polynucleotide base sequence coding for the polypeptide based onthat amino acid sequence, and the step of obtaining a recombinantmicroorganism by introducing that polynucleotide into anothermicroorganism.

The polypeptide of the invention can be purified from the microorganismcontaining that polypeptide in the conventional manner. For example,cells of the microorganism are cultivated in an appropriate medium, andcells are then collected from the culture by centrifugation. The cellsobtained are disrupted using a sonicator, for instance, and the cellresidue is removed by centrifugation, whereby a cell-free extract isobtained. The polypeptide can be purified from this cell-free extract byusing such techniques, either singly or in combination, as salting out(e.g. by ammonium sulfate precipitation, sodium phosphate precipitation,etc.), precipitation using a solvent (protein fractionationprecipitation with acetone, ethanol, etc.), dialysis, gel filtration,ion exchange, reversed phase or like column chromatography, andultrafiltration.

The enzyme activity determination can be carried out by adding thesubstrate 2-chloro-1-(3′-chlorophenyl)ethanone (1 mM) and the coenzymeNADPH (0.25 mM) and the enzyme to 100 mM phosphate buffer (pH 6.5)containing 0.3% (v/v) of dimethyl sulfoxide and measuring the decrementin absorbance at a wavelength of 340 nm at 30° C.

As the polypeptide of the invention, there may be mentioned, forexample, polypeptides having the following physical and chemicalproperties (1) to (4):

(1) activity: acting on 2-chloro-1-(3′-chlorophenyl)ethanone with NADPHor NADH as a coenzyme, to form (R)-2-chloro-1-(3′-chlorophenyl)ethanol;

(2) optimum pH for activity: 5.0 to 6.0;

(3) optimum temperature for activity: 40° C. to 50° C.;

(4) molecular weight: about 40,000 as determined by gel filtrationanalysis, about 30,000 as determined by SDS polyacrylamide gelelectrophoresis analysis.

As the polypeptide of the invention, there may further be mentioned, forexample, (a) a polypeptide comprising the amino acid sequence shownunder SEQ ID NO:1 in the sequence listing or (b) a polypeptidecomprising the amino acid sequence shown under SEQ ID NO:1 in thesequence listing or an amino acid sequence resulting from substitution,insertion, deletion or addition of at least one amino acid residue inthe amino acid sequence shown under SEQ ID NO:1 in the sequence listingand having activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol.

Polypeptides comprising an amino acid sequence derived from the aminoacid sequence shown under SEQ ID NO:1 in the sequence listing resultingfrom substitution, insertion, deletion or addition of at least one aminoacid can be prepared by the conventional method described in CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc., 1989), andthe like. So long as they have activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol, such polypeptides are includedwithin the definition of the polypeptide of the invention.

While any polynucleotide coding for the above polypeptide can be used asthe polynucleotide of the invention, there may be mentioned, forexample, (c) the polynucleotide comprising the base sequence shown underSEQ ID NO:2 in the sequence listing or (d) a polynucleotide capable ofhybridizing with a polynucleotide comprising the base sequencecomplementary to the base sequence shown under SEQ ID NO:2 in thesequence listing under stringent conditions and coding for a polypeptidehaving activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol.

The polynucleotide capable of hybridizing with a polynucleotidecomprising the base sequence complementary to the base sequence shownunder SEQ ID NO:2 in the sequence listing under stringent conditionsmeans a polynucleotide obtained by colony hybridization, plaquehybridization, or southern hybridization, for instance, using apolynucleotide comprising the base sequence complementary to the basesequence shown under SEQ ID NO:2 in the sequence listing as a probe.More specifically, there may be mentioned a polynucleotide that can beidentified after carrying out hybridization using a filter with colony-or plaque-derived polynucleotides immobilized thereon, in the presenceof 0.7 to 1.0 M NaCl at 65° C., and washing the filter with 0.1 to 2×SSCsolution (the composition of 1×SSC solution comprising 150 mM sodiumchloride and 15 mM sodium citrate) at 65° C. The hybridization can becarried out as described in Molecular Cloning, A laboratory manual,second edition (Cold Spring Harbor Laboratory Press, 1989), and thelike.

As the polynucleotide capable of hybridizing, there may be mentioned,specifically, polynucleotides having at least 60%, preferably at least80%, more preferably at least 90%, still more preferably at least 95%,most preferably at least 99%, homology in sequence to the polynucleotideshown under SEQ ID NO:2 in the sequence listing and, so long as thepolypeptides encoded have activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol, they are included within thedefinition of the polynucleotide of the invention.

The “homology (%) in sequence” so referred to herein is expressed interms of the value determined by aligning, in an optimum manner, the twopolynucleotides to be compared, determining the number of those sites ofcoincidence in nucleic acid base (e.g. A, T, C, G, U or I) between theboth, dividing the number by the total number of the bases compared, andmultiplying the result by 100.

The sequence homology can be calculated, for example, by using thefollowing tools for sequence analysis: Unix-based GCG Wisconsin Package(Program Manual for the Wisconsin Package, Version 8, September, 1994,Genetics Computer Group, 575 Science Drive Madison, Wis., USA 53711;Rice, P. (1996), Program Manual for EGCG Package, Peter Rice, The SangerCentre, Hinxton Hall, Cambridge, CB10 1RQ, England) and the ExPASy WorldWide Web Server for Molecular Biology (Geneva University Hospital andUniversity of Geneva, Geneva, Switzerland).

The polynucleotide of the invention can be obtained from a microorganismhaving activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol. As such microorganism, theremay be mentioned, for example, yeasts of the genus Rhodotorula, andRhodotorula glutinis var. dairenensis IFO 0415 may be mentioned as aparticularly preferred one.

In the following, an example is described of the method for obtainingthe polynucleotide of the invention from a microorganism having activityin asymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol. This example is not restrictiveof the scope of the invention, however.

First, partial amino acid sequences of the above-mentioned polypeptideafter purification and peptide fragments obtained by digestion of saidpolypeptide with appropriate endopeptidases are determined by the Edmanmethod. Based on this amino acid sequence information, nucleotideprimers are synthesized. Then, the chromosomal DNA of the microorganismto serve as the source of the polynucleotide of the invention isprepared from that microorganism by the conventional method of DNAisolation such as the method described in Current Protocols in MolecularBiology (John Wiley and Sons, Inc., 1989).

Using this chromosomal DNA as a template, PCR (polymerase chainreaction) is carried out using the nucleotide primers mentioned above tothereby amplify part of the polynucleotide coding for the polypeptide.The base sequence of the thus-amplified polynucleotide can be determinedby the dideoxy sequencing method, dideoxy chain termination method, orthe like. For example, this can be carried out using ABI PRISM DyeTerminator Cycle Sequencing Ready Reaction Kit (product of PerkinElmer)and ABI 373A DNA Sequencer (product of PerkinElmer).

Once part of the base sequence of the polynucleotide coding for saidpolypeptide has become clear, the base sequence of the whole can bedetermined, for example, by the i-PCR method (Nucl. Acids Res., 16, 8186(1988)). When the polynucleotide on the chromosomal DNA contains anintron or introns, the base sequence of the intron-free maturepolynucleotide can be determined, for example, by the following method.Thus, first, from a microorganism to serve as the origin of thepolynucleotide, mRNA of the microorganism is prepared by theconventional method of nucleotide isolation such as the method describedin Current Protocols in Molecular Biology (John Wiley and Sons, Inc.,1989). Then, using this mRNA as a template, a mature polynucleotide isamplified by the RT-PCR method (Proc. Natl. Acad. Sci. USA, 85, 8998(1988)) using nucleotide primers respectively having the sequencesaround the 5′ and 3′ ends of said polynucleotide that have been madeclear in advance, and the base sequence of the mature polypeptide isdetermined in the same manner as described above.

As the vector used for introducing the nucleotide of the invention intoa host microorganism and expressing the same in the host microorganism,any of vectors capable of expressing the gene in said nucleotide in anappropriate host microorganism may be used. As such vectors, there maybe mentioned, for example, ones selected from among plasmid vectors,phage vectors and cosmid vectors. Further, shuttle vectors capable ofgene exchange with another host strain may also be used.

Such vectors generally contain such regulatory factors as the lacUV5promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufBpromoter, recA promoter and pL promoter and can be suitably used asexpression vectors containing an expression unit operatively connectedwith the polynucleotide of the invention.

The term “regulatory factors” as used herein means a base sequencecomprising a functional promoter and arbitrary related transcriptionelements (e.g. enhancer, CCAAT box, TATA box, SPI locus).

The phrase “operatively connected” as used herein means that thepolynucleotide is connected with various regulatory elements controllingthe expression thereof, inclusive of a promoter, an enhancer and soforth, so that the whole can operate in host cells and the gene in saidpolynucleotide can be expressed. It is well known to those skilled inthe art that the types and species of the regulatory factors may varyaccording to the host cells.

As the host cells into which the expression vector containing thepolynucleotide of the invention is to be introduced, there may bementioned bacteria, yeasts, fungi, plant cells and animal cells, and thelike. Escherichia coli cells are particularly preferred, however. Theexpression vector containing the polynucleotide of the invention can beintroduced into host cells in the conventional manner. When Escherichiacoli cells are used as the host cells, the expression vector containingthe polynucleotide of the invention can be introduced thereinto by thecalcium chloride method, for instance.

When (R)-2-chloro-1-(3′-chlorophenyl)ethanol is to be produced byasymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone using thepolypeptide of the invention, a coenzyme such as NADPH or NADH isrequired. However, when an enzyme capable of converting the oxidizedcoenzyme to the reduced form (such ability hereinafter referred to as“coenzyme regenerating ability”) is added to the reaction systemtogether with the substrate thereof, namely when the reaction is carriedout using the coenzyme regenerating system in combination with thepolypeptide of the invention, the usage of the expensive coenzyme can bemarkedly reduced.

Usable as the enzyme having coenzyme regenerating ability are, forexample, hydrogenase, formate dehydrogenase, alcohol dehydrogenase,aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase and glucosedehydrogenase. Glucose dehydrogenase is preferably used.

The asymmetric reduction reaction can be carried out by separatelyadding the above-mentioned enzyme having coenzyme regenerating abilityto the reaction system. It is possible to carry out the reactionefficiently by using, as a catalyst, a transformant as transformed withboth the polynucleotide of the invention and a polynucleotide coding fora polypeptide having coenzyme regenerating ability, without separatelypreparing an enzyme having coenzyme regenerating ability and adding thesame to the reaction system.

Such a transformant can be obtained by inserting the polynucleotide ofthe invention and a polynucleotide coding for a polypeptide havingcoenzyme regenerating ability (e.g. glucose dehydrogenase) into one andthe same vector and introducing the resulting recombinant vector intohost cells or, further, by inserting these two polynucleotidesrespectively into two vectors belonging to different incompatible groupsand introducing the resulting two vectors into the same host cells.

The expression vector of the invention contains the above-mentionedpolynucleotide, as described above. As a preferred example of theexpression vector, there may be mentioned a plasmid pNTRG.

As the expression vector, there may also be mentioned one furthercontaining a polynucleotide coding for the above-mentioned polypeptidehaving glucose dehydrogenase activity. The Bacillus megaterium-derivedglucose dehydrogenase is preferred as the polypeptide having glucosedehydrogenase activity. More preferred is an expression vector which isa plasmid pNTRGG1.

The transformant of the invention is obtained by transforming host cellsusing the above expression vector. Escherichia coli cells are preferredas the host cells.

E. coli HB101(pNTRG) and E. coli HB101(pNTRGG1), which are typicaltransformants of the invention, have been internationally deposited, asof Jan. 22, 2002, with the National Institute of Advanced IndustrialScience and Technology International Patent Organism Depositary, Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, under the accession numbersFERM BP-7857 and FERM BP-7858, respectively, under the Budapest Treaty.

The activity of the enzyme having coenzyme regenerating ability in thetransformant can be measured in the conventional manner. For example,the glucose dehydrogenase activity can be determined by adding 0.1 M ofthe substrate glucose, 2 mM of the coenzyme NADP and the enzyme to 1 MTris hydrochloride buffer (pH 8.0) and measuring the increment inabsorbance at a wavelength 340 nm at 25° C.

The production of an optically active alcohol, such as an opticallyactive 1-phenylethanol derivative or a 3-hydroxy ester derivative, usingthe transformant of the invention can be carried out as follows. Thatis, the culture of the above transformant or a processed product thereofis reacted with a carbonyl group-containing compound to obtain anoptically active alcohol.

More specifically, the carbonyl group-containing compound to serve asthe substrate, such a coenzyme as NADP and the culture of thetransformant or a processed product thereof, and the like are firstadded to an appropriate solvent, and the reaction is allowed to proceedwith stirring under pH adjustment.

The transformant can be cultivated using liquid nutrient mediacontaining ordinary carbon sources, nitrogen sources, inorganic salts,organic nutrients and so forth so long as the microorganism can growthereon. The cultivation temperature is preferably 4 to 50° C.

As the processed product of the transformant there may be mentioned, forexample, crude extracts, cultured cells, lyophilized cells,acetone-dried cells, and products derived therefrom by grinding. It isalso possible to use the polypeptide itself or cells as such in a formimmobilized by a method known in the art.

When a transformant producing both the polypeptide of the invention andan enzyme having coenzyme regenerating ability (e.g. glucosedehydrogenase) is used in carrying out the reaction, it is possible tomarkedly reduce the amount of use of the coenzyme by adding, to thereaction system, a substrate (e.g. glucose) for coenzyme regeneration.

As the carbonyl group-containing compound, which is the substrate, theremay be mentioned, for example, 1-phenylethanone derivatives representedby the formula (3):

in the formula, R¹ and R² may be the same or different and eachrepresents a hydrogen or halogen atom or an alkoxy or nitro group, R³represents a hydrogen or halogen atom, a hydroxyl group or an alkylgroup, which may optionally be substituted, or 3-oxo ester derivativesrepresented by the formula (7):

in the formula, R⁴ represents a hydrogen or halogen atom, an azido orbenzyloxy group or an alkyl group, which may optionally be substituted,and R⁵ represents an alkyl or phenyl group. More specifically, there maybe mentioned, for example, 2-chloro-1-(3′-chlorophenyl)ethanone,1-(2′-fluorophenyl)ethanone, ethyl 4-chloroacetoacetate, and the like.

As the optically active alcohol obtainable by the method mentioned abovethere may be mentioned, for example, optically active 1-phenylethanolderivatives represented by the formula (4):

in the formula, R¹, R² and R³ are as defined above, or optically active3-hydroxy ester derivatives represented by the formula (8):

in the formula, R⁴ and R⁵ are as defined above. More specifically, theremay be mentioned, for example, (R)-2-chloro-1-(3′-chlorophenyl)ethanol,(S)-1-(2′-fluorophenyl)ethanol, ethyl (R)-4-chloro-3-hydroxybutyrate,and the like.

As the halogen atom represented by R¹, R², R³ and/or R⁴, there may bementioned, for example, a fluorine, chlorine, bromine or iodine atom.

As the alkoxy group represented by R¹ and/or R², there may be mentionedalkoxy groups containing 1 to 3 carbon atoms, for example, methoxy,ethoxy, propoxy group, etc., with methoxy group being preferred.

As the alkyl group represented by R³, R⁴ and/or R⁵, there may bementioned alkyl groups containing 1 to 8 carbon atoms, for examplemethyl, ethyl, propyl, hexyl, octyl group, etc. Preferred is an alkylgroup containing 1 or 2 carbon atoms.

The alkyl group represented by R³ and/or R⁴ may optionally besubstituted. As the substituent(s), there may be mentioned fluorine,chlorine and bromine atoms, hydroxyl and amino groups, and the like.

The solvent to be used in carrying out the reaction may be an aqueoussolvent or a mixture of an aqueous solvent and an organic solvent. Asthe organic solvent, there may be mentioned, for example, toluene,hexane, diisopropyl ether, n-butyl acetate, ethyl acetate, and the like.

The reaction temperature is 10° C. to 70° C., preferably 20 to 40° C.,and the reaction time is 1 to 100 hours, preferably 10 to 50 hours.During the reaction, the pH of the reaction mixture is maintained at 4to 10, preferably 5 to 8, using an aqueous solution of sodium hydroxide,an aqueous solution of sodium carbonate, for instance.

Furthermore, the reaction can be carried out either batchwise orcontinuously. In the batchwise case, the reaction substrate is added toa charge concentration of 0.1% to 70% (w/v)

The optically active alcohol formed by the reaction can be purified inthe conventional manner. When the optically active alcohol formed by thereaction is (R)-2-chloro-1-(3′-chlorophenyl)ethanol,(S)-1-(2′-fluorophenyl)ethanol or ethyl (R)-4-chloro-3-hydroxybutyrate,for instance, the suspended matter including microbial cells is removedfrom the reaction mixture by centrifugation, filtration or liketreatment according to need, the product is then extracted with such anorganic solvent as ethyl acetate or toluene, and the organic solvent isthen removed under reduced pressure. The product can be further purifiedby subjecting to such a treatment as distillation and/or chromatography.

The quantitation of 2-chloro-1-(3′-chlorophenyl)ethanone and2-chloro-1-(3′-chlorophenyl)ethanol and the determination of the opticalpurity of 2-chloro-1-(3′-chlorophenyl)ethanol can be carried out byhigh-performance liquid column chromatography (column: Daicel ChemicalIndustries' Chiralcel OJ (ID 4.6 mm×250 mm), eluent:n-hexane/isopropanol=39/1, flow rate: 1 ml/min, detection: 210 nm,column temperature: room temperature).

1-(2′-Fluorophenyl)ethanone and 1-(2′-fluorophenyl)ethanol can bequatitated by high-performance liquid column chromatography (column:Nacalai Tesque's COSMOSIL 5C8-MS (ID 4.6 mm×250 mm), eluent:water/acetonitrile=1/1, flow rate: 1 ml/min, detection: 210 nm, columntemperature: room temperature).

The optical purity of 1-(2′-fluorophenyl)ethanol can be measured byhigh-performance liquid column chromatography (column: Daicel ChemicalIndustries' Chiralcel OB (ID 4.6 mm×250 mm), eluent:n-hexane/isopropanol=9/1, flow rate: 0.5 ml/min, detection: 254 nm,column temperature: room temperature).

Ethyl 4-chloroacetoacetate and ethyl 4-chloro-3-hydroxybutyrate can bequantitated by gas chromatography (column: GL Sciences Inc.'s PEG-20MChromosorb WAW DMCS 10% 80/100 mesh (ID 3 mm×1 m), column temperature:150° C., detection: FID).

The optical purity of ethyl 4-chloro-3-hydroxybutyrate can be determinedby high-performance liquid column chromatography (column: DaicelChemical Industries' Chiralcel OB (ID 4.6 mm×250 mm), eluent:n-hexane/isopropanol=9/1, flow rate: 0.8 ml/min, detection: 215 nm,column temperature: room temperature).

In accordance with the present invention, the polypeptide of theinvention can be produced in an efficient manner and a method forproducing various useful optically active alcohols is provided, asdescribed above.

BRIEF DESCRITPION OF THE DRAWINGS

FIG. 1 shows the polynucleotide sequence of the invention and the aminoacid sequence deduced therefrom.

FIG. 2 shows a method for constructing a recombinant plasmid pNTRGG1 andthe structure thereof.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in detail. Theyare, however, by no means limitative of the scope of the invention.

The details of the procedures in the recombinant DNA technology as usedin the following examples are described in the following monographs:Molecular Cloning, 2nd Edition (Cold Spring Harbor Laboratory Press,1989); Current Protocols in Molecular Biology (Greene PublishingAssociates and Wiley-Interscience).

EXAMPLE 1 Enzyme Purification

An enzyme having activity in asymmetrically reducing2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol was singly purified fromRhodotorula glutinis var. dairenensis IFO 0415. Unless otherwisespecified, the purification procedure was carried out at 4° C.

(Cultivation of Rhodotorula glutinis var. dairenensis IFO 0415)

18 L of a liquid medium having the composition specified below wasprepared in a 30 L jar fermenter (product of B.E. Marubishi Co., Ltd.)and subjected to steam sterilization at 120° C. for 20 minutes.

Medium Composition (% Being Indicated as (w/v)):

Glucose  4.0% Yeast extract  0.3% KH₂PO₄  0.7% (NH₄) ₂HPO₄  1.3% NaCl 0.1% MgSO₄.7H₂O  0.08% ZnSO₄.7H₂O 0.006% FeSO₄.7H₂O 0.009% CuSO₄.5H₂O0.0005%  MnSO₄.4 to 6H₂O 0.001% Adekanol LG-109 (product of NOF Corp.) 0.01% Tap water pH 7.0

This medium was inoculated with a culture containing Rhodotorulaglutinis var. dairenensis IFO 0415 by 180 ml which has been preculturedin advance in the same medium, and cultivation was carried out at a rateof stirring of 250 rpm, a rate of aeration of 5.0 NL/min. and atemperature of 30° C., while the pH was adjusted to a level not lowerthan 5.5 (lower limit) by dropwise addition of a 30% (w/w) aqueoussolution of sodium hydroxide. After the lapse of 18 hours, 22 hours and26 hours from the start of cultivation, 655 g of a 55% (w/w) aqueoussolution of glucose was added on each occasion, and cultivation wascarried out for 30 hours.

(Preparation of a Cell-free Extract)

Cells were collected from a 5,600 ml portion of the above culture bycentrifugation and washed with 1,000 ml of 100 mM phosphate buffer (pH8.2). Thus were obtained 1,599 g of wet cells of the strain mentionedabove. The wet cells were suspended in 100 mM phosphate buffer (pH 8.2)to obtain 2,000 ml of a cell suspension. The cells in this suspensionwere disrupted in DYNO-Mill (product of Willy A. Bachofen Company), andthe disruption product was deprived of cell residue by centrifugation toobtain 1,470 ml of cell-free extract.

(Ammonium Sulfate Fractionation)

Ammonium sulfate was added to and dissolved in the cell-free extractobtained in the above manner to attain 45% saturation, and the resultingprecipitate was removed by centrifugation (on that occasion, the pH ofthe cell-free extract was maintained at 7.5 with aqueous ammonia). Whilemaintaining the pH at 7.5 in the same manner as in the above step,ammonium sulfate was further added to and dissolved in the supernatantresulting from centrifugation to attain 60% saturation, and theresulting precipitate was collected by centrifugation. This precipitatewas dissolved in 10 mM phosphate buffer (pH 7.5), and the solution wasdialyzed overnight with the same buffer.

(DEAE-TOYOPEARL Column Chromatography)

The crude enzyme solution obtained in the above manner was applied, forenzyme adsorption, to a DEAE-TOYOPEARL 650 M (product of TosohCorporation) column (250 ml) equilibrated in advance with 10 mMphosphate buffer (pH 7.5). The column was washed with the same buffer,and an active fraction was then eluted at a linear gradient of NaCl(from 0 M to 0.3 M). The active fraction was collected and dialyzedovernight with 10 mM phosphate buffer (pH 7.5).

(Phenyl-TOYOPEARL Column Chromatography)

Ammonium sulfate was dissolved in the crude enzyme solution obtained inthe above manner to a final concentration of 1 M (while maintaining thepH of the crude enzyme solution at 7.5 with aqueous ammonia), and thesolution was applied, for enzyme adsorption, to a Phenyl-TOYOPEARL 650 M(product of Tosoh Corporation) column (100 ml) equilibrated in advancewith 10 mM phosphate buffer (pH 7.5) containing 1 M ammonium sulfate.After washing the column with the same buffer, the active fraction waseluted at a linear gradient of ammonium sulfate (from 1 M to 0 M). Theactive fraction was collected and dialyzed overnight with 10 mMphosphate buffer (pH 7.5).

(Blue Sepharose Column Chromatography)

The crude enzyme solution obtained as described above was applied, forenzyme adsorption, to a Blue Sepharose CL-6B (product of PharmaciaBiotech) column (20 ml) equilibrated in advance with 10 mM phosphatebuffer (pH 7.5). After washing the column with the same buffer, theactive fraction was eluted at a linear gradient of NaCl (from 0 M to 1M). The active fraction was collected and dialyzed overnight with 10 mMphosphate buffer (pH 7.5) to obtain a purified enzyme preparationshowing a single spot in electrophoresis. Hereinafter, this enzyme isreferred to as RRG.

EXAMPLE 2 Determination of Enzyme Properties

The enzyme obtained was investigated as to its enzymatic properties.

Fundamentally, the enzyme activity was measured by adding the substrate2-chloro-1-(3′-chlorophenyl)ethanone (1 mM), the coenzyme NADPH (0.25mM) and the enzyme to 100 mM phosphate buffer (pH 6.5) containing 0.3%(v/v) dimethyl sulfoxide, allowing the reaction to proceed at 30° C. for1 minute, and then measuring the decrease in absorbance at a wavelengthof 340 nm.

(1) Activity:

In the presence of NADPH as a coenzyme, the enzyme acted on2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol with an optical purity of 99.9%ee or more. The enzyme activity measured with NADH as a coenzyme by theabove method was about 7% of the activity obtained by using NADPH as acoenzyme.

(2) Optimum pH for Activity:

Enzyme activity measurements were carried out by the same enzymeactivity measurement method as mentioned above except that the pH wasvaried in the range of 4.0 to 8.0 using, as the buffer, 100 mM phosphatebuffer containing dimethyl sulfoxide and 100 mM acetate buffer. As aresult, the optimum pH for the action on2-chloro-1-(3′-chlorophenyl)ethanone was found to be 5.0 to 6.0.

(3) Optimum Temperature for Activity:

Enzyme activity measurements were carried out by the same enzymeactivity measurement method as mentioned above except that thetemperature was varied from 20° C. to 60° C. As a result, the optimumtemperature for the action on 2-chloro-1-(3′-chlorophenyl)ethanone wasfound to be 40° C. to 50° C.

(4) Molecular Weight:

The purified enzyme was subjected to gel filtration chromatographyanalysis on Superdex 200 HR 10/30 (product of Amersham PharmaciaBiotech) using 50 mM phosphate buffer (pH 7.0) containing 150 mM sodiumchloride at an eluent. As a result, this enzyme was found to have amolecular weight of about 40,000 as calculated from its relativeretention time as compared with standard proteins. The subunit molecularweight of the enzyme was calculated from the mobility values relative tostandard proteins as obtained by SDS-polyacrylamide gel electrophoresis.The subunit molecular weight of the enzyme was about 30,000.

EXAMPLE 3 RRG Gene Cloning

(PCR Primer Preparation)

The purified enzyme RRG obtained in Example 1 was denatured in thepresence of 8 M urea and then digested with Achromobacter-derived lysylendopeptidase (product of Wako Pure Chemical Industries, Ltd.). Theamino acid sequences of the resulting peptide fragments were determinedusing a model ABI 492 protein sequencer (product of PerkinElmer). Basedon these amino acid sequences, two DNA primers (primer 1, SEQ ID NO:3;primer 2, SEQ ID NO:4) were synthesized in the conventional manner.

(RRG Gene Amplification by PCR)

Chromosomal DNA was extracted from cultured cells of Rhodotorulaglutinis var. dairenensis IFO 0415 by the method of Visser et al. (Appl.Microbiol. Biotechnol., 53, 415 (2000)). Then, using the DNA primersprepared as described above, PCR was carried out with the chromosomalDNA obtained as a template. A DNA fragment, about 600 bp in size andsupposed to be part of the RRG gene, was amplified (PCR was carried outusing TaKaRa Ex Taq (product of Takara Shuzo Co., Ltd.) as DNApolymerase under the reaction conditions described in the manualattached thereto). This DNA fragment was cloned in the plasmid pT7BlueT-Vector (product of Novagen), and the base sequence thereof wasconfirmed using the ABI PRISM Dye Terminator Cycle Sequencing ReadyReaction Kit (product of PerkinElmer) and the ABI 373A DNA Sequencer(product of PerkinElmer).

(Full-length Sequence Determination of the RRG Gene by i-PCR)

The chromosomal DNA of Rhodotorula glutinis var. dairenensis IFO 0415was completely digested with the restriction enzyme SphI, and the DNAfragment mixture obtained was subjected to intramolecular cyclizationusing T4 ligase. Using the cyclization product as a template, thefull-length base sequence of the RRG gene on the chromosomal DNA wasdetermined by the i-PCR technique (Nucl. Acids Res., 16, 8186 (1988))based on the partial base sequence information about the RRG gene asrevealed in the above paragraph (PCR was carried out using TaKaRa LA PCRKit Ver. 2 (product of Takara Shuzo Co., Ltd.)) under the conditionsdescribed in the manual attached thereto. The base sequencedetermination was made as described hereinabove.). The base sequencedetermined is shown under SEQ ID NO:9 in the sequence listing and inFIG. 1. In FIG. 1, the underlined portion is thought to be the sectionencoding the RRG gene in the base sequence, and the other portions to beintrons. The amino acid sequence encoded by the underlined base sequenceportion is shown below the base sequence. Comparison of this amino acidsequence with the partial amino acid sequences of the lysylendopeptidase-digested fragments revealed that the partial amino acidsequences of the purified RRG were all found in this amino acidsequence. The underlined portions in the amino acid sequence shown inFIG. 1 are the portions showing coincidence with the partial amino acidsequences of the purified RRG.

EXAMPLE 4 Intron-free RRG Gene Acquisition

Based on the base sequence determined in Example 3, an N-terminal DNAprimer (primer 3, SEQ ID NO:5) resulting from addition of an NdeI siteto the initiation codon moiety of the RRG gene and a C-terminal DNAprimer (primer 4, SEQ ID NO:6) resulting from addition of a terminationcodon (TAA) and an EcoRI site to just behind the 3′ terminus of the samegene were synthesized. Then, the total RNA of Rhodotorula glutinis var.dairenensis IFO 0415 was extracted and purified from cultured cells ofthat strain using RNeasy Maxi Kit (product of QIAGEN). Using this RNA asa template and using the two DNAs prepared previously as primers, theintron-free mature type RRG gene with an NdeI site added to theinitiation codon moiety and a termination codon (TAA) and an EcoRIcleavage site added just behind the 3′ terminus was amplified by theRT-PCR method (Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)) (RT-PCR wascarried out using High Fidelity RNA PCR Kit (product of Takara ShuzoCo., Ltd.) under the reaction conditions described in the manualattached thereto.).

EXAMPLE 5 Construction of an RRG Gene-containing Recombinant Plasmid

The DNA fragment obtained in Example 4 was digested with NdeI and EcoRI,and the resulting fragment was inserted into the plasmid pUCNT (WO94/03613, U.S. Pat. No. 6,083,752) at the NdeI, EcoRI site downstreamfrom the lac promoter to obtain a recombinant plasmid pNTRG.

EXAMPLE 6 Construction of a Recombinant Plasmid Containing Both the RRGGene and the Glucose Dehydrogenase Gene

A double-stranded DNA derived from the Bacillus megaterium IAM1030-derived glucose dehydrogenase (hereinafter referred to as “GDH”)gene by addition of the Escherichia coli-derived Shine-Dalgarno sequence(9 bases) at a site 5 bases upstream of the initiation codon of thatgene and a SacI cleavage site just before that sequence and, further, aBamHI cleavage site just behind the termination codon was obtained inthe following manner. Based on the base sequence information about theGDH gene, an N-terminal DNA primer (primer 5, SEQ ID NO:7) resultingfrom addition of the Escherichia coli-derived Shaine-Dalgarno sequence(9 bases) at 5 bases upstream of the initiation codon of the structuralgene for GDH and further addition of an EcoRI cleavage site just beforethat sequence and a C-terminal DNA primer (primer 6, SEQ ID NO:8)resulting from addition of a SalI site just behind the termination codonof the structural gene for GDH were synthesized in the conventionalmanner. Using these two DNA primers and using the plasmid pGDK1 (Eur. J.Biochem., 186, 389 (1989)) as a template, a double-stranded DNA wassynthesized by PCR. The DNA fragment obtained was digested with EcoRIand SalI, and the digest was inserted into the pNTRG constructed inExample 5 at the EcoRI, SalI site (occurring downstream from the RRGgene) to obtain a recombinant plasmid pNTRGG1. The construction schemefor and the structure of pNTRGG1 are shown in FIG. 2.

EXAMPLE 7 Recombinant Escherichia coli Production

The recombinant plasmids pNTRG and pNTRGG1 obtained in Examples 5 and 6were used to transform Escherichia coli HB101 (product of Takara ShuzoCo., Ltd.) to obtain transformant Escherichia coli strains HB101 (pNTRG)and HB101 (pNTRGG1), respectively. The thus-obtained transformantsEscherichia coli HB101 (pNTRG) and HB101 (pNTRGG1) have been deposited,as of Jan. 22, 2002, with the National Institute of Advanced IndustrialScience and Technology International Patent Organism Depositary, Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, under the accession numbersFERM BP-7857 and FERM BP-7858, respectively.

EXAMPLE 8 RRG Expression in Recombinant Escherichia coli Species

The recombinant Escherichia coli strain HB101 (pNTRG) obtained inExample 7 was cultivated in 2×YT medium containing 120 μg/ml ofampicillin. Cells were collected and then suspended in 100 mM phosphatebuffer (pH 6.5) and disrupted using a model UH-50 ultrasonic homogenizer(product of SMT Co., Ltd.) to obtain a cell-free extract. The RRGactivity of this cell-free extract was determined in the followingmanner. The RRG activity was determined by adding the substrate2-chloro-1-(3′-chlorophenyl)ethanone (1 mM), the coenzyme NADPH (0.25mM) and the enzyme to 100 mM phosphate buffer (pH 6.5) containing 0.3%(v/v) of dimethyl sulfoxide, and measuring the decrease in absorbance ata wavelength of 340 nm at 30° C. The enzyme activity oxidizing 1 μmol ofNADPH to NADP per minute under these reaction conditions was defined as1 unit. The thus-measured RRG activity in the cell-free extract wasexpressed in terms of specific activity and compared with that of thevector plasmid-harboring transformant. Similarly, the RRG activity inthe cell-free extract from Rhodotorula glutinis var. dairenensis IFO0415 as prepared in the same manner as in Example 1 was determined forcomparison. The results of those determinations are shown in Table 1. Adistinct increase in RRG activity was observed in Escherichia coli HB101(pNTRG) as compared with Escherichia coli HB101 (pUCNT) harboring thevector plasmid alone, and the specific activity was about 150 timeshigher as compared with Rhodotorula glutinis var. dairenensis IFO 0415.

TABLE 1 RRG specific activity Microbial strain (U/mg) E. coli HB101(pUCNT) <0.01 E. coli HB101 (pNTRG) 12.4 Rhodotorula glutinis var.dairenensis IF00415 0.08

EXAMPLE 9 Simultaneous Expression of RRG and GDH in RecombinantEscherichia coli Strains

The recombinant Escherichia coli strain HB101 (pNTRGG1) obtained inExample 7 was treated in the same manner as in Example 8, and thecell-free extract obtained was assayed for GDH activity in the followingmanner. The GDH activity was measured by adding the substrate glucose(0.1 M), the coenzyme NADP (2 mM) and the enzyme to 1 MTris-hydrochloride buffer (pH 8.0) and measuring the increase inabsorbance at a wavelength of 340 nm at 25° C. The enzyme activityreducing 1 μmol of NADP to NADPH per minute under these reactionconditions was defined as 1 unit. The RRG activity was also measured inthe same manner as in Example 8. The thus-measured RRG and GDHactivities of the cell-free extract were each expressed in terms ofspecific activity and compared with the results with Escherichia coliHB101 (pNTRG) and the transformant HB101 (pUCNT) harboring the vectorplasmid alone. The comparative results are shown in Table 2. WithEscherichia coli HB101 (pNTRGG1), distinct increases in RRG activity andGDH activity were found as compared with the transformant Escherichiacoli HB101 (pUCNT) harboring the vector plasmid alone.

TABLE 2 RRG specific GDH specific activity activity Microbial strain(U/mg) (U/mg) E. coli HB101 (pUCNT) <0.01 <0.01 E. coli HB101 (pNTRG)12.4 <0.01 E. coli HB101 (pNTRGG1) 7.56 128

EXAMPLE 10 Synthesis of (R)-2-chloro-1-(3′-chlorophenyl)ethanol from2-chloro-1-(3′-chlorophenyl)ethanone using the Recombinant Escherichiacoli Strain Introduced with the RRG Gene

The culture of the recombinant Escherichia coli HB101(pNTRG) as obtainedin Example 8 was subjected to ultrasonic cell disruption using SONIFIRE250 (product of BRANSON). To 20 ml of this cell disruption fluid, therewere added 2,000 U of glucose dehydrogenase (product of AmanoPharmaceutical Co., Ltd.), 3 g of glucose, 2 mg of NADP and 2 g of2-chloro-1-(3′-chlorophenyl)ethanone. This reaction mixture was stirredat 30° C. for 18 hours while adjusting the pH to 6.5 by addition of 5 Msodium hydroxide. After completion of the reaction, this reactionmixture was extracted with toluene, the solvent was then removed, andthe extract was analyzed. 2-Chloro-1-(3′-chlorophenyl)ethanol wasobtained in 96% yield. The 2-chloro-1-(3′-chlorophenyl)ethanol formed onthat occasion was the R form with an optical purity of 99.9% ee.

The quantitation of 2-chloro-1-(3′-chlorophenyl)ethanone and of2-chloro-1-(3′-chlorophenyl)ethanol and the optical purity measurementof 2-chloro-1-(3′-chlorophenyl)ethanol were carried out byhigh-performance liquid column chromatography (column: Daicel ChemicalIndustries' Chiralcel OJ (ID 4.6 mm×250 mm), eluent:n-hexane/isopropanol=39/1, flow rate: 1 ml/min, detection: 210 nm,column temperature: room temperature).

EXAMPLE 11 Synthesis of (R)-2-chloro-1-(3′-chlorophenyl)ethanol from2-chloro-1-(3′-chlorophenyl)ethanone using the Recombinant Escherichiacoli Strain for Simultaneous Expression of RRG and Glucose Dehydrogenase

The culture of the recombinant Escherichia coli HB101 (pNTRGG1) asobtained in Example 9 was subjected to ultrasonic cell disruption usingSONIFIRE 250 (product of BRANSON). To 20 ml of this cell disruptionfluid, there were added 3 g of glucose, 2 mg of NADP and 2 g of2-chloro-1-(3′-chlorophenyl)ethanone. This reaction mixture was stirredat 30° C. for 24 hours while adjusting the pH to 6.5 by dropwiseaddition of 5 M sodium hydroxide. After completion of the reaction, thisreaction mixture was extracted with toluene, the solvent was thenremoved, and the extract was analyzed in the same manner as in Example10. 2-Chloro-1-(3′-chlorophenyl)ethanol was obtained in 93% yield. The2-chloro-1-(3′-chlorophenyl)ethanol formed on that occasion was the Rform with an optical purity of 99.9% ee.

EXAMPLE 12 Synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate from ethyl4-chloroacetoacetate using the Recombinant Escherichia coli Strain forSimultaneous Expression of RRG and Glucose Dehydrogenase

The culture of the recombinant Escherichia coli HB101 (pNTRGG1) asobtained in Example 9 was subjected to ultrasonic cell disruption usingSONIFIRE 250 (product of BRANSON). To 20 ml of this cell disruptionfluid, there were added 4 g of glucose and 3 mg of NADP. While stirringthis reaction mixture at 30° C. and adjusting the pH to 6.5 by dropwiseaddition of 5 M sodium hydroxide, a total of 2 g of ethyl4-chloroacetoacetate was added to the mixture continuously at a rate of0.2 g per hour. After completion of the addition, the reaction wasfurther allowed to proceed for 12 hours. After completion of thereaction, this reaction mixture was extracted with ethyl acetate, thesolvent was then removed, and the extract was analyzed. Ethyl4-chloro-3-hydroxybutyrate was obtained in 98% yield. The ethyl4-chloro-3-hydroxybutyrate formed on that occasion was the R form withan optical purity of 99% ee or more.

The quantitation of ethyl 4-chloroacetoacetate and of ethyl4-chloro-3-hydroxybutyrate was carried out by gas chromatography(column: GL Sciences Inc.'s PEG-20M Chromosorb WAW DMCS 10% 80/100 mesh(ID 3 mm×1 m), column temperature: 150° C., detection: FID). The opticalpurity of ethyl 4-chloro-3-hydroxybutyratre was determined byhigh-performance liquid column chromatography (column: Daicel ChemicalIndustries' Chiralcel OB (ID 4.6 mm×250 mm), eluent:n-hexane/isopropanol=9/1, flow rate: 0.8 ml/min, detection: 215 nm,column temperature: room temperature).

EXAMPLE 13 Synthesis of (S)-1-(2′-fluorophenyl)ethanol from1-(2′-fluorophenyl)ethanone using the Recombinant Escherichia coliStrain for Simultaneous Expression of RRG and Glucose Dehydrogenase

The culture of the recombinant Escherichia coli HB101(pNTRGG1) asobtained in Example 9 was subjected to ultrasonic cell disruption usingSONIFIRE 250 (product of BRANSON). To 100 ml of this cell disruptionfluid, there were added 15 g of glucose, 5 g of1-(2′-fluorophenyl)ethanone and 15 mg of NADP. This reaction mixture wasstirred at 30° C. for 24 hours while adjusting the pH to 6.5 with 5 Msodium hydroxide. After completion of the reaction, this reactionmixture was extracted with ethyl acetate, the solvent was then removed,and the extract was distilled (54° C./1 mm Hg) to obtain 4.1 g of1-(2′-fluorophenyl)ethanol as a colorless oil. Its specific rotationshowed a value of [α] (25,D)=−45.3 (c=0.794, methanol) and it was the Sform with an optical purity of 99.9% ee.

¹H-NMR (CDCl₃) δ ppm: 1.52 (d, 3H), 1.97 (br, 1H), 5.20 (q, 1H), 6.99 to7.51 (m, 4H)

The quantitation of 1-(2′-fluorophenyl)ethanone and of1-(2′-fluorophenyl)ethanol was carried out by high-performance liquidcolumn chromatography (column: Nacalai Tesque's COSMOSIL 5C8-MS (ID 4.6mm×250 mm), eluent: water/acetonitrile=1/1, flow rate: 1 ml/min,detection; 210 nm, column temperature: room temperature). The opticalpurity of 1-(2′-fluorophenyl)ethanol was determined by high-performanceliquid column chromatography (column: Daicel Chemical Industries'Chiralcel OB (ID 4.6 mm×250 mm), eluent: n-hexane/isopropanol=9/1, flowrate: 0.5 ml/min, detection: 254 nm, column temperature: roomtemperature).

EXAMPLE 14 Substrate Specificity Features of RRG

RRG was examined for reducing activities against various carbonylcompounds. Thus, various carbonyl compounds specified in Table 3 wereused as substrates in lieu of 2-chloro-1-(3′-chlorophenyl)ethanone andactivity measurements were carried out under the basic reactionconditions for the RRG activity measurement in Example 2. Themeasurement results were expressed in terms of relative values with thereducing activity found with 2-chloro-1-(3′-chlorophenyl)ethanone as thesubstrate being taken as 100%. They are shown in Table 3.

TABLE 3 Substrate specificity features of the reductase RRG Reactionsubstrate % Activity 2-chloro-1-(3′-chlorophenyl) 100 ethanone2-acetylpyridine 60 3-acetylpyridine 28 4-acetylpyridine 441-benyl-3-pyrolidinone 4 m-hydroxyacetophenone 16 m-nitroacetophenone 35p-chloroacetophenone 5 4-fluoroacetophenone 30 3,4-dimethoxyacetophenone46 α,α,α-trifluoroacetophenon 83 2-hydroxyacetophenone 34 propiophenone14 n-butyrophenone 8 1-phenyl-2-butanone 20 benzoin 9 trans-calcone 22ethyl benzoylylacetate 15 acetone 11 2-butanone 10 methyl n-propylketone12 2-hexanone 13 2-heptanone 11 diethyl ketone 17 chloroacetone 6hydroxyacetone 16 4-hydroxy-2-butanone 11 diacetyl 4 acetylacetone 234-methyl-2-pentanone 10 cyclopropyl methyl ketone 3 cyclopentanone 9camphorquinone 33 tetrahydrofuran-2,4-dione 14 isophorone 1dihydro-4,4-dimethyl-furandio 238 methyl 2-oxocyclopentane 11carboxylate ethyl 3-oxocyclopentanecarboxylate 32 methyl pyrucvate 208ethly pyruvate 247 methyl acetoacetate 14 ethyl acetoacetate 35tert-butyl acetoacetate 1 ethyl 2-methylacetoacetate 12 ethyl2-chloroacetoacetate 111 methyl 2-eteneacetoacetate 5 methyl2-oxooctanoate 3 methyl 4-chloroacetoacetate 81 ethyl4-chloroacetoacetate 12 n-octyl 4-chloroacetoacetate 72 ethyl4-bromoacetoacetate 33 ethyl 4-azideacetoacetate 11 ethyl4-hydroxyacetoacetate 22 ethyl 4-benzyloxyacetoacetate 58 ethyl4-acetoxyacetoacetate 46 benzyl acetoacetate 30 ethyl2-chloro-3-oxo-3-phen 34 propionate benzaldehyde 252-pyridinecarbaldehyde 45 pyridine-4-aldehyde 55 o-chlorobenzaldehyde 24m-chlorobenzaldehyde 45 p-chlorobenzaldehyde 11 o-nitrobenza1dehyde 31m-nitrobenzaldehyde 64 p-nitrobenzaldhyde 162 propionaldehyde 4n-butylaldhyde 20 n-hexylaldehyde 10 DL-glyceraldehyde 13-phenylpropionaldehyde 10 methyl glyoxal 13 glutaraldehyde 22-keto-n-butyric acid 7 oxalacetic acid 24 levulinic acid 31

INDUSTRIAL APPLICABILITY

As a result of gene cloning of a polypeptide having activity inasymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone to form(R)-2-chloro-1-(3′-chlorophenyl)ethanol and analysis of the nucleotidesequence thereof, it has become possible to obtain a transformant highlycapable of producing the polypeptide. It has also become possible toobtain a transformant capable of producing the polypeptide and glucosedehydrogenase simultaneously at high levels. Further, it has becomepossible to synthesize various optically active alcohols from thecorresponding carbonyl compounds with good efficiency by using saidtransformant.

1. An isolated polynucleotide encoding a polypeptide, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:1.
 2. Anisolated polynucleotide wherein: the polynucleotide comprises SEQ IDNO:2, or the polynucleotide is capable of hybridizing under stringentconditions with the nucleotide sequence complementary to the full lengthof SEQ ID NO:2 and encoding a polypeptide having activity inasymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone representedby the formula (1):

to form (R)-2-chloro-1-(3′-chlorophenyl)ethanol represented by theformula (2):

wherein said stringent conditions involve washing a filter with 0.1 to2×SSC solution at a temperature of 65° C.
 3. An expression vectorcomprising the polynucleotide according to claim
 2. 4. The expressionvector according to claim 3, wherein the expression vector is a plasmidpNTRG.
 5. The expression vector according to claim 3, further comprisinga polynucleotide encoding a polypeptide having glucose dehydrogenaseactivity.
 6. The expression vector according to claim 5, wherein thepolypeptide having glucose dehydrogenase activity is a Bacillusmegaterium-derived glucose dehydrogenase.
 7. The expression vectoraccording to claim 6, wherein the expression vector is a plasmidpNTRGG1.
 8. A transformant, resulting from transforming an isolated hostcell with the expression vector according to claim
 3. 9. Thetransformant according to claim 8, wherein the host cell is Escherichiacoli (E. coli).
 10. The transformant according to claim 9, wherein thetransformant is E. coli HB101(pNTRG) (FERM BP-7857).
 11. Thetransformant according to claim 9, wherein the transformant is E. coliHB101(pNTRGG1) (FERM BP-7858).
 12. The isolated polynucleotide accordingto claim 2, wherein the polypeptide is derived from a microorganismbelonging to the genus Rhodotorula.
 13. The isolated polynucleotideaccording to claim 12, wherein the microorganism belonging to the genusRhodotorula is Rhodotorula glutinis var. dairenensis IFO
 0415. 14. Amethod of producing an optically active 1-phenylethanol derivativerepresented by the formula (4), comprising reacting the culture of thetransformant according to claim 8, or a processed product thereof with acarbonyl group-containing compound represented by formula (3):

wherein, R¹ and R² may be the same or different and each represents ahydrogen or halogen atom or an alkoxy or nitro group, R³ represents ahydrogen or halogen atom, a hydroxyl group or an alkyl group, which mayoptionally be substituted, the formula (4) being:

wherein, R¹, R² and R³ are as defined above.
 15. The method according toclaim 14, wherein the carbonyl group-containing compound is2-chloro-1-(3'-chlorophenyl)ethanone represented by the formula (1):

and the optically active alcohol is(R)-2-chloro-1-(3'-chlorophenyl)ethanol represented by the formula (2):


16. The method according to claim 14, wherein the carbonylgroup-containing compound is 1-(2'-fluorophenyl)ethanone represented bythe formula (5):

and the optically active alcohol is (S)-1-(2'-fluorophenyl)ethanolrepresented by the formula (6):


17. A method for producing optically active 3-hydroxyester derivativerepresented by formula (8), comprising reacting the culture of thetransformant according to claim 8, or a processed product thereof with acarbonyl group-containing compound represented by formula (7):

wherein, R⁴ represents a hydrogen or halogen atom, an azido or benzyloxygroup or an alkyl group, which may optionally be substituted, and R⁵represents an alkyl or phenyl group, the general formula (8) being:

wherein, R⁴ and R⁵ are as defined above.
 18. the method according toclaim 17, wherein the carbonyl group-containing compound is ethyl4-chloroacetoacetate represented by the formula (9):

and the optically active alcohol is ethyl (R)-4-chloro-3-hydroxybutyraterepresented by the formula (10):